Selective expansion of high- or low-avidity cytotoxic T lymphocytes and efficacy for adoptive immunotherapy.

The conventional approach to cytotoxic T-lymphocyte (CTL) induction uses maximal antigen concentration with the intent of eliciting more CTL. However, the efficacy of this approach has not been systematically explored with regard to the quality of the CTLs elicited or their in vivo functionality. Here, we show that a diametrically opposite approach elicits CTLs that are much more effective at clearing virus. CTLs specific for a defined peptide epitope were selectively expanded with various concentrations of peptide antigen. CTLs generated with exceedingly low-dose peptide lysed targets sensitized with > 100-fold less peptide than CTLs generated with high-dose peptide. Differences in expression of T-cell antigen receptors or a number of other accessory molecules did not account for the functional differences. Further, high-avidity CTLs adoptively transferred into severe combined immunodeficient mice were 100- to 1000-fold more effective at viral clearance than the low-avidity CTLs, despite the fact that all CTL lines lysed virus-infected targets in vitro. Thus, the quality of CTLs is as important as the quantity of CTLs for adoptive immunotherapy, and the ability to kill virally infected targets in vitro is not predictive of in vivo efficacy, whereas the determinant density requirement described here is predictive. Application of these principles may be critical in developing effective adoptive cellular immunotherapy for viral infections and cancer.     

Definition of TCR recognition sites on Ld-tum complexes

The P911 variant of the P815 mastocytoma was shown by Lurquinet al. (Cell 58:293,1989) to elicit rapid tumor rejection ina syngeneic host. This rejection was mediated by L^d-restrictedcytotoxic T lymphocytes (CTL) for which targets could be sensitizedby the synthetic peptide designated tum^– (P91A^–.12–24).In a previous study, T cell clones specific for L^d-tum^–complexes displayed very restricted TCR usage and a characteristicTCR motif in the V CDR3 region, predicted to interact with peptide.However, in contrast to the majority of L^d peptide Uganda thatare nonamers, the tum^– peptide is a 13-mer and its sequencedoes not fit the Ld binding motif. Thus, to define shorter versionsof the tum^– 13-mer and residues involved in TCR recognition,nonamer derivatives were synthesized and compared in severaldifferent binding and functional assays. From these comparisons,the peptide TQNHRALDL was found to be the optimal nonamer. CTLrecognition of Ala-substituted analogues of this peptide indicatedthat the Hls and Arg residues at positions 4 and 5 are importantfor TCR contact We propose that these basic residues of thetum^– peptide interact with the previously defined acidicresidues in the CDR3 region of several TCR known to recognizeLd-tum^– complexes.     

Differentiation-dependent differences in murine T cell susceptibility to negative regulation by the lung

A large number of viral infections are contracted via the respiratory route. Thus, an effective immune response at this site is of vital importance. Past studies in murine models analyzing a number of viruses have reported that CD8(+) effector T cells entering the lung after respiratory infection exhibit significant functional inactivation. The impaired function in these cells has been proposed to be the result of infection-induced changes in the lung; however, we have found that loss of function can occur in effector CD8(+) T cells present in the lung, even in the absence of infection. This functional inactivation takes place within 48 hours of entry into the lung, and is seen only in effector cells residing in the lung parenchyma, and not the airway. In this study, we have extended our findings to show that functional impairment of these effector cells is not initiated by bone marrow-derived cells, and is independent of proliferation in the lung tissue. Of critical importance, we have also determined that the susceptibility to functional inactivation is a common property shared by most effector cells. Finally, we show that the susceptibility to loss of function is actively regulated throughout differentiation. Although naive CD8(+) T cells, like effector cells, are negatively regulated as a result of residence in the lung, memory cells exhibit profound resistance to functional inactivation. The selective resistance of CD8(+) memory cells may allow the host to limit damage during the effector phase while retaining a protective response that can effectively limit subsequent infection.     

Perception of multiple-breath inspiratory resistive loads in males and females

Resistive load magnitude estimation (ME) was measured over multiple breaths in male and female subjects. It was hypothesized that multiple breaths against a range of resistive loads would result in a change in the perceived load magnitude as a function of load magnitude and the number of inspiratory efforts. It was further hypothesized that males and females would differ in their perceptual response to sustained breathing against inspiratory resistive loads. The subjects were tested in a sound isolated room and respired through a non-rebreathing valve, the inspiratory port connected to the loading manifold. The subject inspired to a peak airflow target for each breath. Each R load was presented for 10 continuous breaths. The load was estimated at breath 1, 5, and 10 using a modified Borg scale. Each 10-breath load presentation was presented in a randomized block. There was no significant group difference between the ME for breath 1 and 10 for small R loads, but a significant group difference for large R loads. The ME for males did not change between breath 1 and 10 for the small load magnitudes, but decreased with large loads. The ME for the 10th breath of the large R load was greater than the 1st breath for females. Males estimated the large R load on the 1st breath the same as females but the ME on the 10th breath was significantly less for males compared to females. These results demonstrate that magnitude estimation of large resistive loads with a sustained 10-breath trial elicits significant increases in females, but significantly decreased in males. The increase in ME may represent increased respiratory discomfort for females and the decrease habituation in males.     

Loss of function in virus-specific lung effector T cells is independent of infection

Recently, several studies, including those with respiratory syncytial virus, mouse pneumovirus, and simian virus 5, have reported that virus-specific CD8+ effector cells entering the lung as a result of respiratory infection undergo significant loss of function. The impaired function in these cells has been proposed to be the result of infection-induced changes in the lung. Although virus-specific effects may contribute to regulation of T cells in the lung, the findings from this study provide evidence that the basal lung environment is sufficient to promote loss of function in effector cells. Loss of function occurs within 48 h of entry into the lung and is most evident in cells residing in the lung parenchyma. These findings suggest an additional paradigm for the immunoregulation of effector cells that enter the lung as a result of virus infection.     

Supraoptimal Peptide–Major Histocompatibility Complex Causes a Decrease in Bcl-2 Levels and Allows Tumor Necrosis Factor α Receptor II–mediated Apoptosis of Cytotoxic T Lymphocytes

Cytotoxic T lymphocytes (CTLs) are primary mediators of viral clearance, but high viral burden can result in deletion of antigen-specific CTLs. We previously reported a potential mechanism for this deletion: tumor necrosis factor (TNF)-α–mediated apoptosis resulting from stimulation with supraoptimal peptide–major histocompatibility complex. Here, we show that although death is mediated by TNF-α and its receptor (TNF-RII), surprisingly neither the antigen dose dependence of TNF-α production nor that of TNF-RII expression can account for the dose dependence of apoptosis. Rather, a previously unrecognized effect of supraoptimal antigen in markedly decreasing levels of the antiapoptotic protein Bcl-2 was discovered and is likely to account for the gain in susceptibility or competence to sustain the death signal through TNF-RII. This decrease requires a signal through the TCR, not just through TNF-RII. Although death mediated by TNF-RII is not as widely studied as that mediated by TNF-RI, we show here that it is also dependent on proteolytic cleavage by caspases and triggered by a brief initial encounter with antigen. These results suggest that determinant density can regulate the immune response by altering the sensitivity of CTLs to the apoptotic effects of TNF-α by decreasing Bcl-2 levels.     

An abrupt and concordant initiation of apoptosis: antigen-dependent death of CD8+ CTL

The ability of CD8+ cytotoxic T lymphocytes (CTL) to clear viral infections may be limited when high avidity CTL encounter supra-optimal antigen density on antigen-presenting cells (APC) and undergo antigen-dependent apoptosis of CTL (ADAC). Previously, we have shown ADAC in CD8+ populations to be Fas independent, TNF-alpha receptor 2 (TNFR2) mediated, caspase dependent, and accompanied by a decrease in Bcl-2. We now employ flow cytometry to follow ADAC within individual CD8+ cells to demonstrate that the intense TCR signal induced in high avidity CTL by supra-optimal antigen density results 8 - 16 h later in a caspase-independent TNFR2 down-modulation that is directly related to the stimulating APC antigen density and concludes in a rapid onset of apoptosis by 18 - 24 h. Individual CTL undergoing apoptosis exhibit a dramatic and concurrent: (1) positive staining with Annexin V and propidium iodide; (2) transformation to a smaller cell size characteristic of apoptosis; and (3) a nearly complete loss of Bcl-2, c-IAP1, and TRAF2. We conclude that the antigen-dependent apoptosis of CD8+ CTL occurs when a tandem TCR/TNFR2 signal initiates an abrupt and concordant onset of multiple apoptotic events.     

Increased sensitivity to antigen in high avidity CD8(+) T cells results from augmented membrane proximal T-cell receptor signal transduction

The functional avidity of a cytotoxic T lymphocyte (CTL) is known to be a critical determinant of the efficacy with which it clears pathogens. High avidity cells, which are by definition highly sensitive to peptide antigen, are superior for elimination of viruses and tumours. Our studies have established the ability of T cells to undergo avidity modulation as a result of antigen encounter. High and low avidity cells established in this manner exhibit significant differences in the amount of peptide required to elicit effector function. However, how signalling is regulated in these cells as it relates to the control of peptide sensitivity remains to be defined. To address this question, we compared T-cell receptor (TCR) signal transduction events in high and low avidity CTL generated from OT-I(rag2-) TCR transgenic mice. Our data suggest that divergent signalling is initiated at the TCR-associated CD3ζ, with low avidity CTL requiring higher amounts of pMHC to achieve threshold levels of phosphorylated CD3ζ compared with high avidity CTL. Further, this difference is transduced further downstream to mitogen-activated protein kinase and Ca(2+) signalling pathways. These results suggest that regulated control of the initiation of TCR signalling in high versus low avidity cells determines the amount of peptide required for T-cell activation.